Viral reservoir eradication has become a leading subject in the field of HIV research. A plethora of 26 antiretrovirals (ARVs) are available to efficiently treat infection. Living with HIV has become a chronic disease in which viral load can be controlled in over ninety percent of patients. Yet, the health, social, and economical burden of the disease are unprecedented. Social stigma, serious side effects accumulated during long-life treatments, and a tremendous cost to the health system, estimated at near half a million dollars throughout the life of a patient, highlight the need of a cure for HIV. Early in the courseof infection the virus becomes latent within resting cells and remains in a state in which no protein or nucleic acids are expressed, and no new viral particles are made. This reservoir of latentlyinfected cells is now seen as the primary barrier standing in the way of HIV eradication. Even in the presence of efficient cART, over 60 years are required to completely eliminate the reservoir, and alternative approaches to cure HIV are needed. Future efforts to eradicate HIV will likely be based on the so-called shock and kill approach, where the virus is first reactivatd from its latent reservoirs with latency-reversing-agents (LRAs), and a second therapy eliminates reactivated cells. Ex-vivo HIV reactivation assays with autologous virus have recently demonstrated the inability to eliminate reactivated cells in physiological settings. Several factor can explain the partial resistance to CTL-mediated killing, and also preventing death normally induced by the viral CPE. These factors include the low level of expression of viral antigens in reactivated cells and the intrinsic properties of HIV that allow the virus to escape the CTL immune response. In this Phase II program, we propose an HIV-specific immune-based approach to facilitate the killing of infected cells. In a previous Phase I award, we successfully identified small-molecule inhibitors of the Nefmediated MHC-I downmodulation. We characterized molecules that specifically block downmodulation of MHC-I in HIV infected cells without interfering with its surface levels in uninfected cells, and without affecting other unrelated host proteins. These inhibitors prevent the appearance of infected cells with low levels of sMHC-I, which cannot efficiently present viral antigens and are partially resistant to CTL killing. We propose to use these inhibitors to overcome the activity of Nef and facilitate the killng of latently infected cells after pharmacological reactivation with LRAs. The specific aims to optimize inhibitors through medicinal chemistry and identify a preclinical candidate are presented below: Specific Aim 1. Lead optimization of inhibitors of MHC-I downmodulation. Two lead series will be subjected to multiple iterative cycles of lead optimization to improve potency, specificity, metabolic stability, solubility, permeability and CYP profiles, with the goal to identfy a preclinical candidate. Specific Aim 2. Validation of advanced candidates with in vitro, ex-vivo and in vivo assays. Advanced molecules in one lead series will be used to test their effect after addition of CTLs to HIV replicating cells. Inhibitors will also be evaluated with and in-vivo model of HIV latency (humanized BLT mice) to determine whether they diminish the size of the viral reservoir and the frequency of viral rebound after LRA treatment. Specific Aim 3. Preliminary determination of m.o.a. Studies aimed at understanding the mechanism of action of these inhibitors will be carried out. Putative interactions with host partners will be interrogated and those that can be specifically blocked by the MHC-I inhibitors will be further investigated. Success in this Phase II application will be defined as the milestone identification of a preclinicl candidate with adequate PK properties in rats, predictive of oral use in humans. The preclinical candidate will have been validated in ex-vivo assays with samples of aviremic patients, and with the humanized BLT mouse model of latency. These studies may result in first-in-class inhibitors of a novel family of specific immune activators for use in HIV eradication approaches.